US11931401B2 - Runx3 modified protein for prevention or treatment of cancer - Google Patents

Runx3 modified protein for prevention or treatment of cancer Download PDF

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US11931401B2
US11931401B2 US17/239,289 US202117239289A US11931401B2 US 11931401 B2 US11931401 B2 US 11931401B2 US 202117239289 A US202117239289 A US 202117239289A US 11931401 B2 US11931401 B2 US 11931401B2
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runx3
seq
primer
modified protein
protein
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Suk Chul Bae
Jung Won Lee
You Soub Lee
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Genecraft Inc
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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Definitions

  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a Runx3 modified protein as an active ingredient for prevention or treatment of cancer.
  • the said tumor suppressor gene refers to a nucleotide sequence that can be expressed in a target cell to suppress a tumor phenotype or induce apoptosis.
  • the tumor suppressor genes identified so far include sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, Rb, BRCA1 and BRCA2. Among them, it has been reported that p53 or Rb gene is frequently inhibited in its function in K-Ras mutant cancers. Whether it is possible to treat K-Ras mutant cancer through the repair of the suppressor gene has become a subject of great interest in the field of anticancer agent development research.
  • K-Ras mutant lung adenocarcinoma by recovering the function of p53 gene, which is a representative tumor suppressor gene, but it was not successful because early lung adenocarcinoma was not cured (Feldser, D. M. et al., Nature, 468: 572-575, 2010, Junttila, M. R. et al., Nature, 468: 567-571, 2010).
  • K-Ras mutant lung cancer could not be cured through the recovery of Rb gene function (Walter, D. M. et al. Nature 2019).
  • Runx3 gene As a tumor suppressor gene is inhibited in K-Ras mutant cancers (RUNX3 Protects against Oncogenic KRAS. (2013). Cancer Discovery, 4(1), 14-14), and that the activity of Runx3 gene is inhibited in lung adenocarcinoma caused by the mutation of K-Ras (Lee, K. S., Lee, Y. S., Lee, J. M., Ito, K., Cinghu, S., Kim, J. H., Bae, S. C. Oncogene, 29(23): 3349-61, 2010).
  • Runx3 a transcription factor that binds to DNA, plays a crucial role in lineage determination (Ito, Y., Bae, S. C. & Chuang, L. S. The RUNX family: developmental regulators in cancer. Nat. Rev. Cancer 15, 81-95 (2015)). Deletion of Runx3 in the mouse lung leads to the development of lung adenomas and accelerates the progression to adenocarcinoma (ADCs) induced by K-Ras.
  • ADCs adenocarcinoma
  • the present inventors have completed the present invention by confirming that the modified protein in which the 356 th serine of Runx3 is substituted with alanine has an increased activity of maintaining the complex with Brd2 compared to the wild-type Runx3, and the apoptosis effect is improved in various cancer cell lines compared to the wild-type Runx3.
  • It is an object of the present invention to provide a pharmaceutical composition comprising a Runx3 modified protein as an active ingredient for prevention or treatment of cancer.
  • the present invention provides a pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with a hydrophobic amino acid, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
  • a modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with a hydrophobic amino acid, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
  • the modified protein in which the 356 th serine of Runx3 is substituted with alanine has an increased activity of maintaining the complex with Brd2 by more than 10 times compared to the wild-type Runx3, and the apoptosis effect is improved in various cancer cell lines compared to the wild-type Runx3. Therefore, the modified protein in which the 356 th serine of Runx3 is substituted with an amino acid that cannot be phosphorylated by a kinase of the present invention, the polynucleotide coding thereof, the vector carrying the polynucleotide, or the virus or cell transformed with the vector can be used as a therapeutic agent for various cancers.
  • FIG. 1 is a diagram illustrating the time-dependent formation of a BRD2-RUNX3 complex and the RUNX3 phosphorylation in Ser-356 measured by immunoprecipitation (IP) and immunoblotting (IB), confirming that the binding between the overexpressed Myc-RUNX3-S356A and BRD2 was maintained until 8 hours after the serum stimulation.
  • IP immunoprecipitation
  • IB immunoblotting
  • FIGS. 2 a and 2 b are a sequence and a diagram illustrating the predicted degree of phosphorylation of the wild-type protein in which the 356 th serine of Runx3 is not substituted, confirming that the phosphorylation occurred in the 356 th serine.
  • the sequence shown in FIG. 2 a is SEQ ID NO: 2.
  • FIGS. 2 c and 2 d are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356A, in which the 356 th serine of Runx3 is substituted with alanine, confirming that the phosphorylation did not occur in the 356 th alanine.
  • the sequence shown in FIG. 2 c is SEQ ID NO: 2 with a S356A substitution.
  • FIGS. 2 e and 2 f are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356I, in which the 356 th serine of Runx3 is substituted with isoleucine, confirming that the phosphorylation did not occur in the 356 th isoleucine.
  • the sequence shown in FIG. 2 e is SEQ ID NO: 2 with a S356I substitution.
  • FIGS. 2 g and 2 h are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356L, in which the 356 th serine of Runx3 is substituted with leucine, confirming that the phosphorylation did not occur in the 356 th leucine.
  • the sequence shown in FIG. 2 g is SEQ ID NO: 2 with a S356L substitution.
  • FIGS. 2 i and 2 j are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356V, in which the 356 th serine of Runx3 is substituted with valine, confirming that the phosphorylation did not occur in the 356 th valine.
  • the sequence shown in FIG. 2 i is SEQ ID NO: 2 with a S356V substitution.
  • FIG. 3 is a diagram illustrating the time for the modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase and the wild-type Runx3 protein forms a complex with Brd2 protein.
  • FIGS. 4 a - c are diagrams illustrating the cancer cell death rates in lung cancer, stomach cancer, and pancreatic cancer cell lines when the modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase and the wild-type Runx3 protein were administered.
  • FIG. 5 is a diagram illustrating the percentage of cells killed in the lung cancer cell line and the stomach cancer cell line when the modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase and the wild-type Runx3 protein were administered, confirmed by flow cytometry.
  • the modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase and the wild-type Runx3 protein were administered, confirmed by flow cytometry.
  • FIG. 6 is a diagram confirming the expression of Runx3 protein in the adenovirus introduced with the polynucleotide coding the Runx3 S356A modified protein and the wild-type Runx3 protein.
  • Sequence Listing is submitted as an ASCII text file in the form of the file name “Sequence.txt” ( ⁇ 22 kb), which was created on Apr. 22, 2021, and which is incorporated by reference herein.
  • the present invention provides a pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
  • a modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
  • the cancer is solid cancer.
  • the solid cancer can be one or more selected from the group consisting of lung cancer, pancreatic cancer, liver cancer and stomach cancer, but not always limited thereto.
  • the amino acid that cannot be phosphorylated by a kinase can be one or more selected from the group consisting of alanine (A), isoleucine (I), leucine (L) and valine (V), but not always limited thereto.
  • Runx3 (Runt-related transcription factor 3) gene is one of the Runt family genes consisting of Runx1, Runx2 and Runx3.
  • the Runt family genes play an important role in normal development and oncogenesis, and they function as transcriptional regulators of the Smad family, a downstream factor that mediates TGF- ⁇ and its signaling.
  • Runx1 plays an important role in mammalian hematopoiesis
  • Runx2 plays an important role in bone formation
  • Runx3 is mainly expressed in granular gastric mucosal cells, and plays a role in inhibiting cell differentiation of gastric epithelium.
  • These three genes are located at loci of chromosomes 1p, 6p and 21q, of which Runx3 gene is located at 1p36. 11-1p36.
  • Runx3 locus is one of the sites that are lost in a variety of cancers or affected by hemizygous defects.
  • Runx3 has been found to be inactivated in various types of cancer, and it is gaining spotlight as a new target for the development of anticancer agents.
  • Runx3 is known to act as a tumor suppressor gene that suppresses the formation of cancer, and plays an important role in the restriction-point, which determines the fate of cell division and death, and induces cell division or apoptosis depending on the situation (Lee et al., Nat Commun. 2019; 10(1): Runx3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point).
  • Runx3 kills cancer cells by contributing to determining apoptosis fate at the restriction-point (Lee et al., Nat Commun. 2019; 10(1)).
  • a Runx3 protein refers to a Runt-related transcription factor 3 related to the Runt family expressed by the Runx3 gene.
  • the Runx3 protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 1 or SEQ. ID. NO: 2.
  • the Runx3 protein can be derived from humans or animals.
  • the Runx3 protein can be synthesized by the conventional chemical synthesis method in the art (W. H. Freeman and Co., Proteins; structures and molecular principles, 1983), or can be prepared by the conventional genetic engineering method (Maniatis et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual et al.).
  • the Runx3 protein can be a variant of an amino acid sequence having a different sequence by deletion, insertion or substitution of amino acid residues, or a combination thereof within a range that does not affect the function of the protein. Amino acid exchanges in proteins that do not totally alter the activity of the molecule are informed in the art. In some cases, the amino acid can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation or farnesylation. Accordingly, the present invention can include a peptide having an amino acid sequence substantially identical to that of a protein composed of the amino acid sequence represented by SEQ. ID. NO: 1 or SEQ. ID. NO: 2, and variants or fragments thereof. The substantially identical protein can have homology to the protein of the present invention by 80% or more, particularly 90% or more, and more particularly 95% or more.
  • the vector including the polynucleotide encoding the modified protein in which the 356 th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase can be linear DNA or plasmid DNA.
  • the polynucleotide encoding the Runx3 protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 3 or SEQ. ID. NO: 4.
  • the vector refers to a transport mediator for introducing the polynucleotide encoding the modified protein in which the 356 th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase of the present invention into a subject to be treated, and can include a promoter suitable for expression in a subject to be treated, an enhancer, and a polynucleotide encoding the Runx3 protein, a transcription termination site, and the like.
  • the promoter can be a specific organ and tissue specific promoter, and can include a replication origin so as to proliferate in the organ and tissue.
  • BRD2 (Bromodomain-containing protein 2) is a factor that acts as a signaling mediator in the nucleus. It is widely expressed in mammalian cells, and plays an important role in cell cycle regulation and transcriptional regulation.
  • the BRD2 binds to the acetylated Runx3.
  • the BRD2 is composed of BD1 and BD2.
  • the bromodomain 1 (BD1) of the BRD2 binds to the lysine residues 94 and 171 of Runx3.
  • the bromodomain 2 (BD2) of the BRD2 binds to the lysine residue 5 of the acetylated histone 4, the lysine residue 12 of histone 4, and the lysine residue 14 of histone 3.
  • the complex is formed upon receiving mitogenic stimulation.
  • the complex contributes to the determination of restriction point (R-point).
  • the virus transformed by the vector can be any one selected from the group consisting of retrovirus, adenovirus, herpes simplex virus and lentivirus, but not always limited thereto.
  • the vector containing the polynucleotide it is preferably to contain 0.05 to 500 mg, and more preferably to contain 0.1 to 300 mg.
  • the recombinant virus containing the polynucleotide encoding the modified protein in which the 356 th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase it is preferably to contain 10 3 to 10 12 IU (10 to 10 10 PFU), and more preferably to contain 10 5 to 10 10 IU.
  • the recombinant virus is preferably adenovirus.
  • Adeno-associated virus AAV is unsuitable as a delivery vehicle for cancer treatment because its gene expression rate or expression speed is lower than that of adenovirus.
  • Adenovirus is suitable for the delivery of the modified protein according to the present invention to the human body because the transferred gene is expressed in adenovirus more than 3 weeks faster than in adeno-associated virus (HUMAN GENE THERAPY 15:405-413.), and the phenomenon of lowering the gene transfer efficiency due to the immune response is less in the adenovirus than in the adeno-associated virus (World J Gastroenterol. 2016 Jan. 7; 22(1):326-37).
  • the number of viruses for treatment can be represented by the number of viral particles including the vector genome or the number of infectable viruses. That is, since about 1% of the virus particles are the effective number of viruses that can actually be infected, IU (infection unit) or PFU (plaque forming unit) is used to indicate this.
  • the cell transformed by the vector can be bacterium.
  • the bacterium can be non-pathogenic or non-toxic, and can be Listeria, Shigella, Salmonella , or E. coli .
  • DNA of a gene included in the vector can be mass-replicated or proteins can be mass-produced.
  • the vector according to the present invention can be introduced into cells using a method known in the art.
  • transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells can be used to introduce the vector into cells, but not always limited thereto (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
  • the cells transformed with the vector containing the polynucleotide it is preferably to contain 10 3 to 10 8 cells, and more preferably to contain 10 4 to 10 7 cells.
  • the pharmaceutical composition for prevention or treatment of cancer comprising a modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient of the present invention can be administered parenterally during clinical administration.
  • the effective dose of the composition per 1 kg of body weight is 0.05 to 12.5 mg/kg for the vector, 10 7 to 10 11 virus particles (10 5 to 10 9 IU)/kg for the recombinant virus, and 10 3 to 10 6 cells/kg for the cell.
  • the dose is 0.1 to 10 mg/kg for the vector, 10 8 to 10 10 virus particles (10 6 to 10 8 IU)/kg for the recombinant virus, and 10 2 to 10 5 cells/kg for the cell.
  • the composition can be administered 2 to 3 times a day.
  • the composition as described above is not always limited thereto, and can vary depending on the conditions of a patient and the degree of onset of a disease.
  • the pharmaceutical composition according to the present invention may contain 10 to 95 weight % of a vector containing a Runx3 protein, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector, which is an active ingredient, based on the total weight of the composition.
  • the pharmaceutical composition of the present invention can include, in addition to the active ingredient, one or more effective ingredients having the same or similar function to the active ingredient.
  • the present inventors confirmed that phosphorylation did not occur in the modified protein in which the 356 th amino acid of Runx3 is substituted with another amino acid ( FIGS. 2 a to 2 j ), the modified protein had a complex maintenance activity of more than 10 times compared to that the binding between the wild-type Runx3 and BRD2 was separated after 3 hours since the physical binding of the modified protein in which the 356 th serine of Runx3 is substituted with alanine with BRD2 was maintained for up to 40 hours ( FIG.
  • the modified protein in which the 356 th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, the polynucleotide coding thereof, the vector carrying the polynucleotide, or the virus or cell transformed with the vector can be used as a therapeutic agent for various cancers.
  • HEK293 cells ATCC, Manassas, VA, USA
  • DMEM medium Gibco BRL, Thermo Fisher Scientific, MA, USA, MA
  • penicillin/streptomycin Invitrogen, Carlsbad, CA, USA
  • H460 cells ATCC, Manassas, VA, USA
  • H460 stable cells were maintained in RPMI 1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin (Invitrogen).
  • MKN28 cells ATCC, Manassas, VA, USA
  • PANC1 cells ATCC, Manassas, VA, USA
  • All the cell lines were cultured in a 37° C., 5% CO 2 incubator.
  • Each cell line was cultured in a 10 cm culture dish (5 ⁇ 10 5 cells) for 2 days and then approximately 2 ⁇ 10 6 cells of each cell line was infected with 1 ⁇ 10 8 VP of adenovirus or adenovirus Runx3 or adenovirus Runx3 S356A. After 48 hours, the cell death rate was measured by flow cytometry.
  • Transient transfection was performed in all cell lines using lipofectamine plus reagent and lipofectamine (Invitrogen).
  • Cell lysates were incubated with an appropriate monoclonal or polyclonal antibody (2 ⁇ g of antibody/500 ⁇ g of lysate sample) at 4° C. for 3 hours, followed by incubation with protein G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
  • protein G-Sepharose beads Amersham Pharmacia Biotech, Piscataway, NJ, USA.
  • the lysate was incubated with an appropriate monoclonal or polyclonal antibody (1:1000 ⁇ 1:3000) at 4° C.
  • the antibody targeting RUNX3 (Cat #ab40278) was obtained from Abcam (Cambridge, UK), and the antibody was diluted 1:3000.
  • BRD2 (M01; 1:1000; Cat #H00006046-M01, Abnova, Taipei City, Taiwan) was used for immunoblotting and immunoprecipitation.
  • Example 1 Production of a Modified Protein in which the 356 th Serine of Runx3 Protein is Substituted
  • mutant recombination was performed in the animal cell expression vector pCS4-3flag-RUNX3 using the primer sets listed in Table 1 including EcoRI-XhoI cleavage sites at both ends to induce mutations in the codon encoding the amino acid sequence of the 356 th serine.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 5) substituted Primer-R: att ctc gag tca gta ggg ccg c with alanine. (SEQ. ID. NO: 6) (TCA ⁇ GCA) Primer-M-F: ggc gac cgc gca cct acc c (SEQ. ID. NO: 7) Primer-M-R: g ggt agg tgc gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 9) substituted Primer-R: att ctc gag tca gta ggg ccg c with valine. (SEQ. ID. NO: 10) (TCA ⁇ GTA) Primer-M-F: ggc gac cgc gta cct acc c (SEQ. ID. NO: 11) Primer-M-R: g ggt agg tac gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 13) substituted Primer-R: att ctc gag tca gta ggg ccg c with (SEQ. ID. NO: 14) isoleucine.
  • Primer-M-F ggc gac cgc ata cct acc c (TCA ⁇ ATA) (SEQ. ID. NO: 15)
  • Primer-M-R g ggt agg tat gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 17) substituted Primer-R: att ctc gag tca gta ggg ccg c with leucine. (SEQ. ID. NO: 18) (TCA ⁇ CTA) Primer-M-F: ggc gac cgc cta cct acc c (SEQ. ID. NO: 19) Primer-M-R: g ggt agg tag gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 29) substituted Primer-R: att ctc gag tca gta ggg ccg c with (SEQ. ID. NO: 30) asparagine.
  • Primer-M-F ggc gac cgc aat cct acc c (TCA ⁇ AAT) (SEQ. ID. NO: 31)
  • Primer-M-R g ggt agg att gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 37) substituted Primer-R: att ctc gag tca gta ggg ccg c with (SEQ. ID. NO: 38) glutamine.
  • Primer-M-F ggc gac cgc caa cct acc c (TCA ⁇ CAA) (SEQ. ID. NO: 39)
  • Primer-M-R g ggt agg ttg gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 41) substituted Primer-R: att ctc gag tca gta ggg ccg c with histidine.
  • SEQ. ID. NO: 42 (TCA ⁇ CAC)
  • Primer-M-F ggc gac cgc cac cct acc c
  • Primer-M-R g ggt agg gtg gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 53) substituted Primer-R: att ctc gag tca gta ggg ccg c with (SEQ. ID. NO: 54) phenylalanine.
  • Primer-M-F ggc gac cgc ttc cct acc c (TCA ⁇ TTC) (SEQ. ID. NO: 55)
  • Primer-M-R g ggt agg gaa gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 57) substituted Primer-R: att ctc gag tca gta ggg ccg c with proline. (SEQ. ID. NO: 58) (TCA ⁇ CCA) Primer-M-F: ggc gac cgc cca cct acc c (SEQ. ID. NO: 59) Primer-M-R: g ggt agg tgg gcg gtc gcc (SEQ. ID.
  • Primer-F cgg gaa ttc a atg cgt att ccc gta Serine was (SEQ. ID. NO: 61) substituted Primer-R: att ctc gag tca gta ggg ccg c with (SEQ. ID. NO: 62) tryptophane.
  • Primer-M-F ggc gac cgc tgg cct acc c (TCA ⁇ TGG) (SEQ. ID. NO: 63)
  • Primer-M-R g ggt agg cca gcg gtc gcc (SEQ. ID. NO: 64)
  • pCS4-flag-RUNX3 plasmid was digested with EcoRI/XhoI restriction enzyme and polymerase chain reaction (PCR) was performed with each Primer-F/Primer-M-R (Resulting Insert: F) and Primer-M-F/Primer-R (Resulting Insert: R) using RUNX3 Insert as a template.
  • the PCR product was purified, and the secondary polymerase chain reaction was performed with F and R templates, Primer-F and Primer-R.
  • the RUNX3 S356A PCR product obtained by the secondary polymerase chain reaction was purified.
  • RUNX3 S356A and pCS4-flag-Vector were digested with EcoRI/XhoI restriction enzyme, gel-extracted, ligated, and transfected. Only single colony was cultured in 2 ml and of LB medium, and the cloned plasmid was separated and purified to confirm the nucleotide sequence.
  • the modified protein in which the 356 th amino acid of Runx3 is substituted with alanine blocks the physical binding between Runx3 and CDK4, thereby inhibiting the phosphorylation of the 356 th serine of Runx3 by CDK4.
  • the function of maintaining the anticancer activity of Runx3 is improved.
  • the following experiment was performed to compare the time for the modified protein in which the 356 th amino acid of Runx3 is substituted with alanine to form a complex by binding to Brd2 protein with the wild-type Runx3.
  • the antibody targeting RUNX3 (Cat #ab40278) was obtained from Abcam (Cambridge, UK), and the antibody was diluted 1:3000.
  • BRD2 (M01; 1:1000; Cat #H00006046-M01, Abnova, Taipei City, Taiwan) was used for immunoblotting and immunoprecipitation. Transient transfection was performed in all cell lines using lipofectamine plus reagent and lipofectamine (Invitrogen). Cell lysates were incubated with an appropriate monoclonal or polyclonal antibody (2 ⁇ g of antibody/500 ⁇ g of lysate sample) at 4° C.
  • the wild-type Runx3 bound with Brd2 and formed a complex for up to 8 hours, but the modified protein in which the 356 th serine of Runx3 is substituted with alanine maintained the physical binding with BRD2 for up to 40 hours.
  • the modified protein had a complex maintenance activity of 10 times or more compared to the wild-type, as the binding of the wild-type Runx3 and BRD2 was separated from 3 hours after the binding.
  • Runx3 S356A modified protein improved the maintenance efficacy of the Runx3-Brd2 complex, and the apoptosis ability thereof was improved in various cancer cell lines compared to the wild-type Runx3.
  • each cell line prepared according to Experimental Method 1 above was harvested and processed using FITC-Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, Calif., USA) and propidium iodide DNA staining protocol. Apoptosis and cell cycle were analyzed by flow cytometry on a BD FACS caliber machine (BD Biosciences). All data were analyzed using FlowJo software (https://www.flowjo.com).
  • the modified protein in which the 356 th serine of Runx3 is substituted with alanine increased the apoptosis rate by about 2 times or more in lung cancer cell lines, and further increased the cancer cell death efficacy in stomach cancer and pancreatic cancer cell lines compared to the wild-type Runx3 protein.
  • Runx3 protein was expressed in both the adenovirus into which the polynucleotide encoding the Runx3 S356A modified protein was introduced and the adenovirus into which the polynucleotide encoding the wild-type Runx3 protein was introduced.

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Abstract

In the present invention, it was confirmed that the modified protein in which the 356th serine of Runx3 is substituted with alanine has an increased activity of maintaining the complex with Brd2 by more than 10 times compared to the wild-type Runx3, and the apoptosis effect is improved in various cancer cell lines compared to the wild-type Runx3. Therefore, the modified protein in which the 356th serine of Runx3 is substituted with an amino acid that cannot be phosphorylated by a kinase of the present invention, the polynucleotide coding thereof, the vector carrying the polynucleotide, or the virus or cell transformed with the vector can be used as a therapeutic agent for various cancers.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority from Korean Patent Application No. 10-2020-0049341, filed on Apr. 23, 2020, and Korean Patent Application No. 10-2020-0184526, filed on Dec. 28, 2020, the contents of each of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to a pharmaceutical composition comprising a Runx3 modified protein as an active ingredient for prevention or treatment of cancer.
2. Description of the Related Art
Research on the development of targeted cancer therapy is focused on strategies to control cancer cells by inhibiting the function of an oncogene or activating the function of a tumor suppressor gene. Abnormal activation of K-Ras function by mutation of K-Ras among the oncogenes is known as one of the major causes of human cancer. The mutation of K-Ras is also observed in lung cancer, and it is known that the mutation of K-Ras is observed in about 35% of lung adenocarcinoma. Thus, in order to treat cancer caused by the activation of K-Ras function, studies have been conducted on a method of treating cancer by inhibiting the function of K-Ras. However, a strategy that directly inhibits the function of K-Ras has not been developed as a successful anticancer drug because it causes serious damage to normal cells. Therefore, instead of suppressing the function of an oncogene, a strategy of activating the inhibited function of a tumor suppressor gene is receiving attention. Therefore, instead of a strategy for inhibiting the function of an oncogene, a strategy for activating the inhibited function of a tumor suppressor gene is attracting attention.
The said tumor suppressor gene refers to a nucleotide sequence that can be expressed in a target cell to suppress a tumor phenotype or induce apoptosis. The tumor suppressor genes identified so far include sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, Rb, BRCA1 and BRCA2. Among them, it has been reported that p53 or Rb gene is frequently inhibited in its function in K-Ras mutant cancers. Whether it is possible to treat K-Ras mutant cancer through the repair of the suppressor gene has become a subject of great interest in the field of anticancer agent development research. Accordingly, there have been attempts to treat K-Ras mutant lung adenocarcinoma by recovering the function of p53 gene, which is a representative tumor suppressor gene, but it was not successful because early lung adenocarcinoma was not cured (Feldser, D. M. et al., Nature, 468: 572-575, 2010, Junttila, M. R. et al., Nature, 468: 567-571, 2010). In addition, it was found that K-Ras mutant lung cancer could not be cured through the recovery of Rb gene function (Walter, D. M. et al. Nature 2019). The above results indicate that even if the function of the tumor suppressor gene is simply restored, the therapeutic effect on the already-onset cancer does not appear, because the early stage cancer rapidly develops into a malignant cancer (Berns A., Nature, 468:519-520, 2010). There have been no reports of successful treatment of K-Ras mutant lung cancer through the activation of a tumor suppressor gene.
It has been reported that the function of Runx3 gene as a tumor suppressor gene is inhibited in K-Ras mutant cancers (RUNX3 Protects against Oncogenic KRAS. (2013). Cancer Discovery, 4(1), 14-14), and that the activity of Runx3 gene is inhibited in lung adenocarcinoma caused by the mutation of K-Ras (Lee, K. S., Lee, Y. S., Lee, J. M., Ito, K., Cinghu, S., Kim, J. H., Bae, S. C. Oncogene, 29(23): 3349-61, 2010).
Runx3, a transcription factor that binds to DNA, plays a crucial role in lineage determination (Ito, Y., Bae, S. C. & Chuang, L. S. The RUNX family: developmental regulators in cancer. Nat. Rev. Cancer 15, 81-95 (2015)). Deletion of Runx3 in the mouse lung leads to the development of lung adenomas and accelerates the progression to adenocarcinoma (ADCs) induced by K-Ras.
Thus, the present inventors have completed the present invention by confirming that the modified protein in which the 356th serine of Runx3 is substituted with alanine has an increased activity of maintaining the complex with Brd2 compared to the wild-type Runx3, and the apoptosis effect is improved in various cancer cell lines compared to the wild-type Runx3.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a pharmaceutical composition comprising a Runx3 modified protein as an active ingredient for prevention or treatment of cancer.
To achieve the above object, the present invention provides a pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with a hydrophobic amino acid, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
Advantageous Effect
In the present invention, it was confirmed that the modified protein in which the 356th serine of Runx3 is substituted with alanine has an increased activity of maintaining the complex with Brd2 by more than 10 times compared to the wild-type Runx3, and the apoptosis effect is improved in various cancer cell lines compared to the wild-type Runx3. Therefore, the modified protein in which the 356th serine of Runx3 is substituted with an amino acid that cannot be phosphorylated by a kinase of the present invention, the polynucleotide coding thereof, the vector carrying the polynucleotide, or the virus or cell transformed with the vector can be used as a therapeutic agent for various cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating the time-dependent formation of a BRD2-RUNX3 complex and the RUNX3 phosphorylation in Ser-356 measured by immunoprecipitation (IP) and immunoblotting (IB), confirming that the binding between the overexpressed Myc-RUNX3-S356A and BRD2 was maintained until 8 hours after the serum stimulation.
FIGS. 2 a and 2 b are a sequence and a diagram illustrating the predicted degree of phosphorylation of the wild-type protein in which the 356th serine of Runx3 is not substituted, confirming that the phosphorylation occurred in the 356th serine. The sequence shown in FIG. 2 a is SEQ ID NO: 2.
FIGS. 2 c and 2 d are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356A, in which the 356th serine of Runx3 is substituted with alanine, confirming that the phosphorylation did not occur in the 356th alanine. The sequence shown in FIG. 2 c is SEQ ID NO: 2 with a S356A substitution.
FIGS. 2 e and 2 f are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356I, in which the 356th serine of Runx3 is substituted with isoleucine, confirming that the phosphorylation did not occur in the 356th isoleucine. The sequence shown in FIG. 2 e is SEQ ID NO: 2 with a S356I substitution.
FIGS. 2 g and 2 h are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356L, in which the 356th serine of Runx3 is substituted with leucine, confirming that the phosphorylation did not occur in the 356th leucine. The sequence shown in FIG. 2 g is SEQ ID NO: 2 with a S356L substitution.
FIGS. 2 i and 2 j are a sequence and a diagram illustrating the predicted degree of phosphorylation of S356V, in which the 356th serine of Runx3 is substituted with valine, confirming that the phosphorylation did not occur in the 356th valine. The sequence shown in FIG. 2 i is SEQ ID NO: 2 with a S356V substitution.
FIG. 3 is a diagram illustrating the time for the modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase and the wild-type Runx3 protein forms a complex with Brd2 protein.
FIGS. 4 a-c are diagrams illustrating the cancer cell death rates in lung cancer, stomach cancer, and pancreatic cancer cell lines when the modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase and the wild-type Runx3 protein were administered.
FIG. 5 is a diagram illustrating the percentage of cells killed in the lung cancer cell line and the stomach cancer cell line when the modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase and the wild-type Runx3 protein were administered, confirmed by flow cytometry.
FIG. 6 is a diagram confirming the expression of Runx3 protein in the adenovirus introduced with the polynucleotide coding the Runx3 S356A modified protein and the wild-type Runx3 protein.
 SEQUENCE
The Sequence Listing is submitted as an ASCII text file in the form of the file name “Sequence.txt” (˜22 kb), which was created on Apr. 22, 2021, and which is incorporated by reference herein.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the present invention is described in detail.
The present invention provides a pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
The cancer is solid cancer.
The solid cancer can be one or more selected from the group consisting of lung cancer, pancreatic cancer, liver cancer and stomach cancer, but not always limited thereto.
The amino acid that cannot be phosphorylated by a kinase can be one or more selected from the group consisting of alanine (A), isoleucine (I), leucine (L) and valine (V), but not always limited thereto.
Runx3 (Runt-related transcription factor 3) gene is one of the Runt family genes consisting of Runx1, Runx2 and Runx3. The Runt family genes play an important role in normal development and oncogenesis, and they function as transcriptional regulators of the Smad family, a downstream factor that mediates TGF-β and its signaling. Runx1 plays an important role in mammalian hematopoiesis, Runx2 plays an important role in bone formation, and Runx3 is mainly expressed in granular gastric mucosal cells, and plays a role in inhibiting cell differentiation of gastric epithelium. These three genes are located at loci of chromosomes 1p, 6p and 21q, of which Runx3 gene is located at 1p36. 11-1p36. 13. The Runx3 locus is one of the sites that are lost in a variety of cancers or affected by hemizygous defects. In addition, Runx3 has been found to be inactivated in various types of cancer, and it is gaining spotlight as a new target for the development of anticancer agents. As such, Runx3 is known to act as a tumor suppressor gene that suppresses the formation of cancer, and plays an important role in the restriction-point, which determines the fate of cell division and death, and induces cell division or apoptosis depending on the situation (Lee et al., Nat Commun. 2019; 10(1): Runx3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point). When a K-Ras oncogene mutation occurs in lung epithelial cells, Runx3 kills cancer cells by contributing to determining apoptosis fate at the restriction-point (Lee et al., Nat Commun. 2019; 10(1)).
A Runx3 protein refers to a Runt-related transcription factor 3 related to the Runt family expressed by the Runx3 gene.
The Runx3 protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 1 or SEQ. ID. NO: 2.
The Runx3 protein can be derived from humans or animals.
The Runx3 protein can be synthesized by the conventional chemical synthesis method in the art (W. H. Freeman and Co., Proteins; structures and molecular principles, 1983), or can be prepared by the conventional genetic engineering method (Maniatis et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual et al.).
The Runx3 protein can be a variant of an amino acid sequence having a different sequence by deletion, insertion or substitution of amino acid residues, or a combination thereof within a range that does not affect the function of the protein. Amino acid exchanges in proteins that do not totally alter the activity of the molecule are informed in the art. In some cases, the amino acid can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation or farnesylation. Accordingly, the present invention can include a peptide having an amino acid sequence substantially identical to that of a protein composed of the amino acid sequence represented by SEQ. ID. NO: 1 or SEQ. ID. NO: 2, and variants or fragments thereof. The substantially identical protein can have homology to the protein of the present invention by 80% or more, particularly 90% or more, and more particularly 95% or more.
The vector including the polynucleotide encoding the modified protein in which the 356th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase can be linear DNA or plasmid DNA.
The polynucleotide encoding the Runx3 protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 3 or SEQ. ID. NO: 4.
The vector refers to a transport mediator for introducing the polynucleotide encoding the modified protein in which the 356th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase of the present invention into a subject to be treated, and can include a promoter suitable for expression in a subject to be treated, an enhancer, and a polynucleotide encoding the Runx3 protein, a transcription termination site, and the like. The promoter can be a specific organ and tissue specific promoter, and can include a replication origin so as to proliferate in the organ and tissue.
BRD2 (Bromodomain-containing protein 2) is a factor that acts as a signaling mediator in the nucleus. It is widely expressed in mammalian cells, and plays an important role in cell cycle regulation and transcriptional regulation.
The BRD2 binds to the acetylated Runx3.
The BRD2 is composed of BD1 and BD2.
The bromodomain 1 (BD1) of the BRD2 binds to the lysine residues 94 and 171 of Runx3.
The bromodomain 2 (BD2) of the BRD2 binds to the lysine residue 5 of the acetylated histone 4, the lysine residue 12 of histone 4, and the lysine residue 14 of histone 3.
When the complex is formed, cell death occurs.
In addition, the complex is formed upon receiving mitogenic stimulation.
The complex contributes to the determination of restriction point (R-point).
The virus transformed by the vector can be any one selected from the group consisting of retrovirus, adenovirus, herpes simplex virus and lentivirus, but not always limited thereto.
In the case of the vector containing the polynucleotide, it is preferably to contain 0.05 to 500 mg, and more preferably to contain 0.1 to 300 mg. In the case of the recombinant virus containing the polynucleotide encoding the modified protein in which the 356th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase, it is preferably to contain 103 to 1012 IU (10 to 1010 PFU), and more preferably to contain 105 to 1010 IU.
The recombinant virus is preferably adenovirus. Adeno-associated virus (AAV) is unsuitable as a delivery vehicle for cancer treatment because its gene expression rate or expression speed is lower than that of adenovirus. Adenovirus is suitable for the delivery of the modified protein according to the present invention to the human body because the transferred gene is expressed in adenovirus more than 3 weeks faster than in adeno-associated virus (HUMAN GENE THERAPY 15:405-413.), and the phenomenon of lowering the gene transfer efficiency due to the immune response is less in the adenovirus than in the adeno-associated virus (World J Gastroenterol. 2016 Jan. 7; 22(1):326-37).
The number of viruses for treatment can be represented by the number of viral particles including the vector genome or the number of infectable viruses. That is, since about 1% of the virus particles are the effective number of viruses that can actually be infected, IU (infection unit) or PFU (plaque forming unit) is used to indicate this.
The cell transformed by the vector can be bacterium.
The bacterium can be non-pathogenic or non-toxic, and can be Listeria, Shigella, Salmonella, or E. coli. By introducing the vector into bacteria, DNA of a gene included in the vector can be mass-replicated or proteins can be mass-produced.
The vector according to the present invention can be introduced into cells using a method known in the art. For example, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells can be used to introduce the vector into cells, but not always limited thereto (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
In the case of the cells transformed with the vector containing the polynucleotide, it is preferably to contain 103 to 108 cells, and more preferably to contain 104 to 107 cells.
The pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient of the present invention can be administered parenterally during clinical administration.
The effective dose of the composition per 1 kg of body weight is 0.05 to 12.5 mg/kg for the vector, 107 to 1011 virus particles (105 to 109 IU)/kg for the recombinant virus, and 103 to 106 cells/kg for the cell. Preferably, the dose is 0.1 to 10 mg/kg for the vector, 108 to 1010 virus particles (106 to 108 IU)/kg for the recombinant virus, and 102 to 105 cells/kg for the cell. The composition can be administered 2 to 3 times a day. The composition as described above is not always limited thereto, and can vary depending on the conditions of a patient and the degree of onset of a disease.
The pharmaceutical composition according to the present invention may contain 10 to 95 weight % of a vector containing a Runx3 protein, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector, which is an active ingredient, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention can include, in addition to the active ingredient, one or more effective ingredients having the same or similar function to the active ingredient.
In preferred embodiments of the present invention, the present inventors confirmed that phosphorylation did not occur in the modified protein in which the 356th amino acid of Runx3 is substituted with another amino acid (FIGS. 2 a to 2 j ), the modified protein had a complex maintenance activity of more than 10 times compared to that the binding between the wild-type Runx3 and BRD2 was separated after 3 hours since the physical binding of the modified protein in which the 356th serine of Runx3 is substituted with alanine with BRD2 was maintained for up to 40 hours (FIG. 3 ), and the modified protein in which the 356th serine of Runx3 is substituted with alanine was more effective in killing cancer cells in lung cancer, gastric cancer and pancreatic cancer cell lines compared to the wild-type Runx3 protein (FIGS. 4 a-4 c and 5).
Therefore, the modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, the polynucleotide coding thereof, the vector carrying the polynucleotide, or the virus or cell transformed with the vector can be used as a therapeutic agent for various cancers.
Hereinafter, the present invention will be described in detail by the following examples and experimental examples.
However, the following examples and experimental examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.
<Experimental Methods>
1. Cell Line Preparation
HEK293 cells (ATCC, Manassas, VA, USA) were maintained in DMEM medium (Gibco BRL, Thermo Fisher Scientific, MA, USA, MA) supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).
H460 cells (ATCC, Manassas, VA, USA) and H460 stable cells were maintained in RPMI 1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin (Invitrogen). MKN28 cells (ATCC, Manassas, VA, USA) and PANC1 cells (ATCC, Manassas, VA, USA) were maintained in DMEM medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL) and penicillin/streptomycin (Invitrogen). All the cell lines were cultured in a 37° C., 5% CO2 incubator.
2. Introduction of Runx3 and Runx3 S356A
Each cell line was cultured in a 10 cm culture dish (5×105 cells) for 2 days and then approximately 2×106 cells of each cell line was infected with 1×108 VP of adenovirus or adenovirus Runx3 or adenovirus Runx3 S356A. After 48 hours, the cell death rate was measured by flow cytometry.
3. DNA Transfection, Immunoprecipitation (IP) and Immunoblotting (IB)
Transient transfection was performed in all cell lines using lipofectamine plus reagent and lipofectamine (Invitrogen). Cell lysates were incubated with an appropriate monoclonal or polyclonal antibody (2 μg of antibody/500 μg of lysate sample) at 4° C. for 3 hours, followed by incubation with protein G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For the detection of endogenous proteins at 4° C. for 1 hour, the lysate was incubated with an appropriate monoclonal or polyclonal antibody (1:1000˜1:3000) at 4° C. for 6 to 12 hours, and then protein G-Sepharose beads (Amersham Pharmacia Biotech) were heated at 4° C. for 3 hours. The immune precipitate was digested on an SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked, immunoblotted with an appropriate antibody, treated with ECL solution (Amersham Pharmacia Biotech), and visualized in Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA).
4. Antibody
The antibody targeting RUNX3 (5G4) (Cat #ab40278) was obtained from Abcam (Cambridge, UK), and the antibody was diluted 1:3000. BRD2 (M01; 1:1000; Cat #H00006046-M01, Abnova, Taipei City, Taiwan) was used for immunoblotting and immunoprecipitation.
5. Flow Cytometry
Cells were harvested and processed using FITC-Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) and propidium iodide DNA staining protocol. Apoptosis and cell cycle were analyzed by flow cytometry on a BD FACS caliber machine (BD Biosciences). All data were analyzed using FlowJo software (https://www.flowjo.com).
Example 1: Production of a Modified Protein in which the 356th Serine of Runx3 Protein is Substituted
In the polynucleotide encoding the Runx3 protein registered in Genebank, mutant recombination was performed in the animal cell expression vector pCS4-3flag-RUNX3 using the primer sets listed in Table 1 including EcoRI-XhoI cleavage sites at both ends to induce mutations in the codon encoding the amino acid sequence of the 356th serine.
TABLE 1
Name Primer sequence Characteristics
S356A Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 5) substituted
Primer-R: att ctc gag tca gta ggg ccg c with alanine.
(SEQ. ID. NO: 6) (TCA→GCA)
Primer-M-F: ggc gac cgc gca cct acc c
(SEQ. ID. NO: 7)
Primer-M-R: g ggt agg tgc gcg gtc gcc
(SEQ. ID. NO: 8)
S356V Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 9) substituted
Primer-R: att ctc gag tca gta ggg ccg c with valine.
(SEQ. ID. NO: 10) (TCA→GTA)
Primer-M-F: ggc gac cgc gta cct acc c
(SEQ. ID. NO: 11)
Primer-M-R: g ggt agg tac gcg gtc gcc
(SEQ. ID. NO: 12)
S356I Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 13) substituted
Primer-R: att ctc gag tca gta ggg ccg c with
(SEQ. ID. NO: 14) isoleucine.
Primer-M-F: ggc gac cgc ata cct acc c (TCA→ATA)
(SEQ. ID. NO: 15)
Primer-M-R: g ggt agg tat gcg gtc gcc
(SEQ. ID. NO: 16)
S356L Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 17) substituted
Primer-R: att ctc gag tca gta ggg ccg c with leucine.
(SEQ. ID. NO: 18) (TCA→CTA)
Primer-M-F: ggc gac cgc cta cct acc c
(SEQ. ID. NO: 19)
Primer-M-R: g ggt agg tag gcg gtc gcc
(SEQ. ID. NO: 20)
S356G Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 21) substituted
Primer-R: att ctc gag tca gta ggg ccg c with glycine.
(SEQ. ID. NO: 22) (TCA→GGA)
Primer-M-F: ggc gac cgc gga cct acc c
(SEQ. ID. NO: 23)
Primer-M-R: g ggt agg tcc gcg gtc gcc
(SEQ. ID. NO: 24)
S356R Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 25) substituted
Primer-R: att ctc gag tca gta ggg ccg c with arginine.
(SEQ. ID. NO: 26) (TCA→CGA)
Primer-M-F: ggc gac cgc cga cct acc c
(SEQ. ID. NO: 27)
Primer-M-R: g ggt agg tcg gcg gtc gcc
(SEQ. ID. NO: 28)
S356N Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 29) substituted
Primer-R: att ctc gag tca gta ggg ccg c with
(SEQ. ID. NO: 30) asparagine.
Primer-M-F: ggc gac cgc aat cct acc c (TCA→AAT)
(SEQ. ID. NO: 31)
Primer-M-R: g ggt agg att gcg gtc gcc
(SEQ. ID. NO: 32)
S356C Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 33) substituted
Primer-R: att ctc gag tca gta ggg ccg c with cysteine.
(SEQ. ID. NO: 34) (TCA→TGC)
Primer-M-F: ggc gac cgc tgc cct acc c
(SEQ. ID. NO: 35)
Primer-M-R: g ggt agg gca gcg gtc gcc
(SEQ. ID. NO: 36)
S356Q Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 37) substituted
Primer-R: att ctc gag tca gta ggg ccg c with
(SEQ. ID. NO: 38) glutamine.
Primer-M-F: ggc gac cgc caa cct acc c (TCA→CAA)
(SEQ. ID. NO: 39)
Primer-M-R: g ggt agg ttg gcg gtc gcc
(SEQ. ID. NO: 40)
S356H Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 41) substituted
Primer-R: att ctc gag tca gta ggg ccg c with histidine.
(SEQ. ID. NO: 42) (TCA→CAC)
Primer-M-F: ggc gac cgc cac cct acc c
(SEQ. ID. NO: 43)
Primer-M-R: g ggt agg gtg gcg gtc gcc
(SEQ. ID. NO: 44)
S356K Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 45) substituted
Primer-R: att ctc gag tca gta ggg ccg c with lysine.
(SEQ. ID. NO: 46) (TCA→AAA)
Primer-M-F: ggc gac cgc aaa cct acc c
(SEQ. ID. NO: 47)
Primer-M-R: g ggt agg ttt gcg gtc gcc
(SEQ. ID. NO: 48)
S356M Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 49) substituted
Primer-R: att ctc gag tca gta ggg ccg c with
(SEQ. ID. NO: 50) methionine.
Primer-M-F: ggc gac cgc atg cct acc c (TCA→ATG)
(SEQ. ID. NO: 51)
Primer-M-R: g ggt agg cat gcg gtc gcc
(SEQ. ID. NO: 52)
S356F Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 53) substituted
Primer-R: att ctc gag tca gta ggg ccg c with
(SEQ. ID. NO: 54) phenylalanine.
Primer-M-F: ggc gac cgc ttc cct acc c (TCA→TTC)
(SEQ. ID. NO: 55)
Primer-M-R: g ggt agg gaa gcg gtc gcc
(SEQ. ID. NO: 56)
S356P Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 57) substituted
Primer-R: att ctc gag tca gta ggg ccg c with proline.
(SEQ. ID. NO: 58) (TCA→CCA)
Primer-M-F: ggc gac cgc cca cct acc c
(SEQ. ID. NO: 59)
Primer-M-R: g ggt agg tgg gcg gtc gcc
(SEQ. ID. NO: 60)
S356W Primer-F: cgg gaa ttc a atg cgt att ccc gta Serine was
(SEQ. ID. NO: 61) substituted
Primer-R: att ctc gag tca gta ggg ccg c with
(SEQ. ID. NO: 62) tryptophane.
Primer-M-F: ggc gac cgc tgg cct acc c (TCA→TGG)
(SEQ. ID. NO: 63)
Primer-M-R: g ggt agg cca gcg gtc gcc
(SEQ. ID. NO: 64)
Particularly, pCS4-flag-RUNX3 plasmid was digested with EcoRI/XhoI restriction enzyme and polymerase chain reaction (PCR) was performed with each Primer-F/Primer-M-R (Resulting Insert: F) and Primer-M-F/Primer-R (Resulting Insert: R) using RUNX3 Insert as a template. The PCR product was purified, and the secondary polymerase chain reaction was performed with F and R templates, Primer-F and Primer-R. The RUNX3 S356A PCR product obtained by the secondary polymerase chain reaction was purified. RUNX3 S356A and pCS4-flag-Vector were digested with EcoRI/XhoI restriction enzyme, gel-extracted, ligated, and transfected. Only single colony was cultured in 2 ml and of LB medium, and the cloned plasmid was separated and purified to confirm the nucleotide sequence.
Experimental Example 1: Confirmation of Phosphorylation Inhibitory Effect when the 356th Serine of Runx3 Protein is Substituted
RUNX3 phosphorylation in serine, the 356th amino acid of Runx3 protein, was measured by immunoprecipitation (IP) and immunoblotting (IB) in the same manner as described in Experimental Method 3 above.
As a result, as shown in FIG. 1 , the binding between Myc-RUNX3-S356A and BRD2 was maintained until 8 hours after the serum stimulation. The above results indicate that substitution of serine, the 356th amino acid of Runx3 protein, to alanine inhibited phosphorylation of Runx3, thereby the time for maintaining the binding with BRD2 was increased.
Experimental Example 2: Confirmation of Non-Phosphorylation of 356th Amino Acid of Runx3 Modified Protein
The possibility of phosphorylation of the 356th amino acid of the prepared mutant was verified using a phosphorylation level prediction tool (Phospho.elm:http://phospho.elm.eu.org/pELMB lastSearch.html) and NETPhos 3.1 (cbs.dtu.dk/services/NetPhos/). As a result, as shown in Table 2 and FIGS. 2 a to 2 j , it was confirmed that there was no phosphorylation in the part.
The above results indicate that when the 356th amino acid of Runx3 was substituted with another amino acid, the phosphorylation of Runx3 was suppressed, and thus the time for maintaining the binding with BRD2 was increased.
TABLE 2
Phosphorylation
356th amino acid prediction result
S356 wild type Serine O
S356A Alanine X
S356I Isoleucine X
S356L Leucine X
S356V Valine X
S356C Cysteine X
S356F Phenylalanine X
S356G Glycine X
S356K Lysine X
S356H Histidine X
S356M Methionine X
S356N asparagine X
S356P Proline X
S356Q Glutamine X
S356R arginine X
S356W tryptophan X
Experimental Example 3: Confirmation of Increasing Effect on the Stability of Runx3 Protein by Runx3 S356A Modified Protein
The modified protein in which the 356th amino acid of Runx3 is substituted with alanine blocks the physical binding between Runx3 and CDK4, thereby inhibiting the phosphorylation of the 356th serine of Runx3 by CDK4. By inhibiting the conversion of the Rpa-Rx3-AC complex including Runx3 to the Rpa-Rx3-TR complex, the function of maintaining the anticancer activity of Runx3 is improved. Thus, the following experiment was performed to compare the time for the modified protein in which the 356th amino acid of Runx3 is substituted with alanine to form a complex by binding to Brd2 protein with the wild-type Runx3.
Particularly, the antibody targeting RUNX3 (5G4) (Cat #ab40278) was obtained from Abcam (Cambridge, UK), and the antibody was diluted 1:3000. BRD2 (M01; 1:1000; Cat #H00006046-M01, Abnova, Taipei City, Taiwan) was used for immunoblotting and immunoprecipitation. Transient transfection was performed in all cell lines using lipofectamine plus reagent and lipofectamine (Invitrogen). Cell lysates were incubated with an appropriate monoclonal or polyclonal antibody (2 μg of antibody/500 μg of lysate sample) at 4° C. for 3 hours, followed by incubation with protein G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, N.J., USA). For the detection of endogenous proteins at 4° C. for 1 hour, the lysate was incubated with an appropriate monoclonal or polyclonal antibody (1:1000˜1:3000) at 4° C. for 6 to 12 hours, and then protein G-Sepharose beads (Amersham Pharmacia Biotech) were heated at 4° C. for 3 hours. The immune precipitate was digested on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked, immunoblotted with an appropriate antibody, treated with ECL solution (Amersham Pharmacia Biotech), and visualized in Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA).
As a result, as shown in FIGS. 2 a-2 j , the wild-type Runx3 bound with Brd2 and formed a complex for up to 8 hours, but the modified protein in which the 356th serine of Runx3 is substituted with alanine maintained the physical binding with BRD2 for up to 40 hours. Thus, it was confirmed that the modified protein had a complex maintenance activity of 10 times or more compared to the wild-type, as the binding of the wild-type Runx3 and BRD2 was separated from 3 hours after the binding.
Experimental Example 4: Confirmation of Apoptotic Effect of Runx3 S356A Modified Protein More Effective than Wild-Type Runx3 on Solid Cancer Cell Lines
It was confirmed through flow cytometry that the Runx3 S356A modified protein improved the maintenance efficacy of the Runx3-Brd2 complex, and the apoptosis ability thereof was improved in various cancer cell lines compared to the wild-type Runx3.
Particularly, each cell line prepared according to Experimental Method 1 above was harvested and processed using FITC-Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, Calif., USA) and propidium iodide DNA staining protocol. Apoptosis and cell cycle were analyzed by flow cytometry on a BD FACS caliber machine (BD Biosciences). All data were analyzed using FlowJo software (https://www.flowjo.com).
As a result, as shown in FIGS. 3 and 4 a-4 c, the modified protein in which the 356th serine of Runx3 is substituted with alanine increased the apoptosis rate by about 2 times or more in lung cancer cell lines, and further increased the cancer cell death efficacy in stomach cancer and pancreatic cancer cell lines compared to the wild-type Runx3 protein.
Experimental Example 5: Measurement of Runx3 Expression Level in Runx3 S356A Modified Protein and Wild-Type Runx3 Protein
Western blotting was performed as described in Experimental Method 3 to confirm the expression of Runx3 protein in adenovirus expressing the Runx3 S356A modified protein and the wild-type Runx3.
As a result, as shown in FIG. 5 , it was confirmed that the Runx3 protein was expressed in both the adenovirus into which the polynucleotide encoding the Runx3 S356A modified protein was introduced and the adenovirus into which the polynucleotide encoding the wild-type Runx3 protein was introduced.

Claims (11)

What is claimed is:
1. A method for treating cancer comprising administering a modified protein, a polynucleotide encoding the modified protein, a vector carrying the polynucleotide, or a virus or cell transformed with the vector, in a pharmaceutically effective amount to a subject in need thereof,
wherein the modified protein is a Runx3 (Runt-related transcription factor 3) protein in which the 356th serine according to SEQ ID NO: 2 is substituted with an amino acid that cannot be phosphorylated by a kinase, and wherein the cancer is lung, stomach, or pancreatic cancer.
2. The method for treating cancer according to claim 1, wherein the amino acid that cannot be phosphorylated by a kinase is one or more selected from the group consisting of alanine (A), isoleucine (I), leucine (L) and valine (V).
3. The method for treating cancer according to claim 1, wherein SEQ ID NO: 2 is encoded by a polynucleotide represented by SEQ ID NO: 4.
4. The method for treating cancer according to claim 1, wherein the vector is linear DNA or plasmid DNA.
5. The method for treating cancer according to claim 1, wherein the virus is any one selected from the group consisting of retrovirus, adenovirus, herpes simplex virus and lentivirus.
6. The method for treating cancer according to claim 1, wherein the cell is bacterium.
7. The method for treating cancer according to claim 6, wherein the bacterium is Listeria, Shigella, Salmonella, or E. coli.
8. The method for treating cancer according to claim 1, wherein the modified protein binds BRD2 at least 10 times longer than wild-type Runx3 in vitro.
9. The method for treating cancer according to claim 1, wherein the cancer is lung cancer.
10. The method for treating cancer according to claim 9, wherein the amino acid that cannot be phosphorylated by a kinase is one or more selected from the group consisting of alanine (A), isoleucine (I), leucine (L) and valine (V).
11. The method for treating cancer according to claim 9, wherein the amino acid that cannot be phosphorylated is alanine (A).
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